Approximately 2/3rd of the world population is at risk of infection by at least one of the 35 insect-borne flaviviruses known to cause disease in humans. There are currently few vaccines and no therapeutics available to treat patients infected by flaviviruses such as Dengue, Zika, and West Nile viruses despite the severe morbidity and mortality they cause globally each year. The development of improved vaccines and therapeutics to prevent and treat flavivirus infections requires improved knowledge of the molecular mechanisms these serious human pathogens use to replicate their genomes. RNA capping of flavivirus genomes has received increasing attention over the last decade as an antiviral drug target due to its critical roles in maintaining viral RNA stability, controlling viral protein translation, and innate immune evasion. There is, however, not much known about how flavivirus RNAs are capped during infection. Therefore, this proposal will define how flaviviruses cap their RNA genomes during infection and evaluate how capping affects innate immune evasion. 1) The NS5 RNA guanylyltransferase is a novel flavivirus enzyme with no structural or sequence similarities to any other known nucleotidyltransferase enzyme. It is currently unknown how this important viral enzyme functions, which is a critical gap in our understanding of flavivirus RNA replication. We are using a combination mutagenesis and viral replication experiments to define the active site of the flavivirus NS5 guanylyltransferase, providing the first in-depth characterization of this unique viral replication enzyme. 2) The 5' untranslated region (UTR) of the flavivirus genome contains conserved sequence and structural elements known to be involved in RNA replication, but their role in RNA capping has never been assessed. We will use a series of mutated 5' UTR RNAs to test the specificity of NS5-mediated RNA capping to define how 5' UTR terminal sequences and the stem-loop A structure affect binding and capping efficiency. 3) Degradation of flavivirus genomes by the cellular RNA decay pathway results in the inhibition of RNA decay and RNAi pathways, altering the immune response to infection. We wish to test the intriguing hypothesis that viral capping efficiency may be strategically regulated by viruses to produce non-coding RNAs that alter infection dynamics. Interestingly, we have recently found that uncapped viral RNAs are incorporated into virus particles and may comprise up to a third of viral RNAs in an infected cell. This surprisingly high level of uncapped RNA is likely processed by RNA decay factors and leads to high levels of a small sfRNA which has been shown previously to antagonize RNAi and interferon responses (among other things). We will examine, therefore, how NS5 capping efficiency affects viral RNA production, the fate of uncapped viral RNAs in cells, and how the products of these uncapped RNAs influence the dynamics of a flavivirus infection. Overall, this project will significantly advance our understanding of flavivirus replication mechanisms that we can exploit for antiviral and vaccine development, and will provide critical new information about how flaviviruses cause disease.